Although all cells of one organism contain more or less the same genetic information, genes are turned on and others are turned off at different locations and times during the life cycle of an organism. The intricate pattern of gene regulation involves molecular signals that act on DNA sequences encoding protein products. Such a DNA sequence that facilitates the transcription of a particular gene is called a promoter. The promoter is the main determinant for the initiation of transcription and modulation of levels and timing of gene expression.
Certain promoters are either silent or active at very low background levels in normal tissues but highly active in tumors. A number of promoters can be included within this rather heterogeneous group:    (1) Promoters which are specific for the malignant process but which show no particular tissue specificity—so-called “cancer specific promoters”.    (2) Promoters of genes which encode onco-fetal antigens and which have very well-defined patterns of tissue specificity—so-called “tumor-type specific promoters”.    (3) Promoters responsive to patho-physiological conditions which predominate in tumor areas (e.g. hypoxia responsive promoters),    (4) Promoters which are specific to the tumor vascular endothelium.Cancer Specific Promoters
Certain genes are up regulated as part of the malignant phenotype in a range of tumours in a tissue non-specific manner. The promoters of such genes potentially represent powerful targets because they may provide a means of targeting therapeutic genes to a variety of malignant tissues. This raises the possibility of constructing generic cancer specific vectors, which will be applicable across a broad range of oncological practice, without the need to tailor design of the promoters used on an individual, patient by patient, basis.
Telomerase is not expressed in normal tissues (except germ cells and stem cells), but is abnormally reactivated in all major cancer types (Buys, C. H. 2000, N. Engl. J. Med. 342, 1282-1283.; Shay, J. W. 1998, Cancer J. Sci. Am. 4 Suppl 1, S26-34). Telomerase enables tumour cells to maintain telomere length, thus circumventing the process of senescence. Many cancer cell lines can be passaged indefinitely and are considered immortal, whereas normal cells senescence after a set number of population doublings called the Hayflick limit. Telomerase expression has been reported in the vast majority of cancers and its promoter has been utilised for cancer therapeutics both by RNA interference approaches as well as gene suiciding approaches (Plumb et al., 2001, Oncogene 20, 7797-7803; Xing et al., 2008, Cancer Biol. Ther. 7, 1839-1848). However, It should be borne in mind that there still exists the possibility of germ line or stem cell toxicity with this strategy as telomerase activity is reported in such cellular systems (Wright et al., 1996, Dev. Genet. 18, 173-179). This limits the applications of telomerase promoter for targeted cancer therapeutics as it can elicit its effects within untransformed or normal cellular machinery too.
Tumour-Type Specific Promoters
Onco-fetal antigens are proteins which are expressed during fetal life as a part of normal development and are silenced in the adult. These proteins can be re-expressed in certain malignant conditions. The classical examples are carcinoembryonic antigen (CEA), which is expressed by a number of adenocarcinomas including colorectal, breast and lung cancers, and alpha fetoprotein (AFP), which is expressed by hepatocellular carcinomas and malignant testicular teratomas (Harrington et al., 2000, Adv. Drug Deliv. Rev. 44, 167-184). The promoters of these genes have been characterized and their essential elements have been identified.
A number of authors have used the CEA promoter to the drive expression of either reporter or therapeutic genes in gastric, lung and colorectal tumour systems (Cao et al., 1999, Gene Ther. 6, 83-90.). In most of these studies, adeno virus (AV) vectors have been used with the CEA promoter controlling the expression of a suicide gene. Such vectors have been shown to confer selective gene expression both in vitro and in vivo after intra-peritoneal (Lan et al., 1997, Cancer Res. 57, 4279-4284) or intra-tumoral injection (Brand et al., 1998, Gene Ther. 5, 1363-1371). A similar body of work exists for the treatment of hepatonma with AFP-regulated gene therapy systems (Su et al., 1997, Proc. Natl. Acad, Sci. U.S.A 94, 13891-13896). Impressive in vitro viral replication and toxicity were seen with hepatoma cell lines and these data translated to tumour responses in subcutaneous hepatomas (but not non-hepatomas) in nude mice (Arbuthnot et al., 1996, Hum. Gene Ther. 7, 1503-1514; Kaneko et al., 1995, Cancer Res. 0.55, 5283-5287).
Promoters Specific to Cancer Pathophysiology
Cellular hypoxia induces a stress response in which the expression of many genes is increased. Not surprisingly, a common underlying theme to the functions of these genes is to promote processes which will relieve hypoxia, such as short-term measures like shifting the emphasis of cellular respiration towards the glycolytic pathway and longer-term responses like increasing erythropoiesis and angiogenesis (Dachs and Stratford, 1996, Br. J. Cancer. Suppl. 27, S126-132), The genes that mediate these adaptive responses phosphoglycerate kinase 1, erythropoietin (Epo) and vascular endothelial growth factor (VEGF) genes, respectively all have promoters which contain cis-acting hypoxia response elements (HRE) which are capable of binding either hypoxia inducible factor 1 (HIF-1) or other related proteins (Maxwell et al., 1997, Proc. Natl. Acad. Sci. U.S.A 94, 8104-8109; Pugh et al., 1991, Proc. Natl. Acad. Sci, U.S.A 88, 10553-10557; Wang and Semenza, 1993, J. Biol. Chem. 268, 21513-21518). In normal healthy tissue, hypoxia is rare, perhaps with the exception of some cartilaginous tissues. However, hypoxia, often to a profound degree, is a common feature in many solid tumours and is thought to play a significant role in the resistance of cancer to ionising radiation and cytotoxic chemotherapy. The use of promoter elements responsive to tissue hypoxia in gene therapy strategies offers the prospect of turning the tables on the tumour and using this treatment-resistant pathophysiological state to drive the expression of therapeutic genes (Dachs et al., 1997, at. Med. 3, 515-520).
Endothelium-Specific Promoters
In the last 20 years, there has been an increasing appreciation of the central role played by the tumour vasculature in the progression and dissemination of malignant disease (Folkman, 1996, Eur. J. Cancer Oxf. Engl. 1990 32A, 2534-2539). It is clear that in order to increase beyond a certain size limit, a tumour must recruit an adequate blood supply. It does this, at least in part, by stimulating the ingress of new blood vessels by secreting angiogenic factors such as VEGF or by causing them to be secreted by stromal cells. As a consequence of these observations, the tumour neo-vasculature has become a legitimate target of cancer gene therapy (Harris, 1997, Lancet 349 Suppl 2, SII13-15). The attraction of destroying the tumour neovasculature is that it offers the prospect of killing a large number of dependent tumour cells through a form of anatomical bystander effect. In recent years, a number of genes, which are up-regulated in the proliferating endothelium of tumour blood vessels, have been identified. Jaggar et al., (1997, Hum. Gene Ther. 8, 2239-2247) reported that proximal elements from both the VEGF responsive kinase insert domain receptor (KDR) and the E-selectin promoters are capable of directing endothelial cell specific gene expression. Walton et al., 1998 used an adenoviral vector in which a luciferase reporter was under the transcriptional control of the E-selectin promoter. High levels of reporter gene expression were reported in endothelial cells in a fashion that was upregulated on exposure to either TNF-α or tumour-conditioned medium. There was little gene expression in non-endothelial tissues. Another gene that has been shown to be overexpressed in tumour vasculature is endoglin (a member of the transforming growth factor beta receptor complex). Graulich et al., (1999, Gene 227, 55-62) have identified its promoter and demonstrated that it shows significant tissue specificity and greater strength than the SV40 promoter in endothelial cells (HUVEC, HMVEC and ECV304 cells) as compared to fibroblasts or epithelial cells.
A number of promoters have already been investigated with positive results: for example, α-fetoprotein (AFP) promoter to target hepatocellular carcinoma (Ido et al., 2001, Cancer Res. 61, 3016-3021), prostate-specific antigen (PSA) to target prostate cancer (Latham et al., 2000a, Cancer Res. 60, 334-341) and so on.
Several examples showed that the use of enhancers in conjunction with specific promoters in viral constructs might be beneficial. Placing a 1455-bp PSA-enhancer sequence upstream of either the PSA or the glandular kallicrein promoter (hKLK2) increased the expression of the marker gene in the PSA-positive prostate cancer cell line LNCaP by 20-fold. Tandem duplication of the PSA enhancer increased expression to 50-fold while retaining tissue specificity (Ido et al., 2001, Cancer Res. 61, 3016-3021; Latham et al., 2000, Cancer Res. 60, 334-341).
Transcriptional Gene Silencing (TGS) refers to using siRNA to target the enhancer or promoter regions of genes thereby leading to the downregulation of their expression. These siRNAs have been shown to repress gene expression by DNA methylation or Histone methylation (Morris, 2009 RNA Biol. 6, 242-247; Morris et al., 2004 Science 305, 1289-1292; Morris et al. 2008, PLoS Genet. 4, e1000258; Napoli et al., 2009, EMBO J. 28, 1708-1719).
HPV is central to the development of cervical neoplasia, with HPV 16 being the most prevalent in squamous cell carcinoma and HPV 18 most prevalent in adenocarcinoma (Alani and Münger, 1998, J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 16, 330-337). The E6 and E7 oncoproteins are the main transforming genes of oncogenic strains of HPV. The HPV E7 protein acts primarily by binding to and inactivating the retinoblastoma (Rb) tumor suppressor gene product (Smotkin and Wettstein, 1986, Proc. Natl. Acad. Sci. U.S.A. 83, 4680-4684). The E6 proteins of oncogenic HPV subtypes binds to and inactivate the p53 tumor suppressor gene product. Both E6 & E7 are governed by a common promoter/enhancer region, so targeting this common region by Transcriptional Gene Silencing (TGS) could be used as more potent tool to tackle HPV-16 associated cervical cancer as this would result in the down regulation of both onco-proteins avoiding the need to separately design the siRNA for these sequence (Palanichamy et al., 2010, Mol. Cancer Ther. 9, 2114-2122). Additionally, the effect of TGS is long lasting and genetically inherited to daughter cells. This therapeutic approach of gene silencing could be further honed and made tumour specific by utilizing PLAP promoter/enhancer system to drive the expression of shRNA targeting the LCR of these E6/E7 onco-proteins.
In case of HPV, since both the oncogenes (E6/E7) are driven by a common enhancer and promoter, targeting of the enhancer with siRNA could cause a potent decrease in the expression of the oncogenes E6 and E7 leading to apoptosis of the malignant cells.
c-Myc is a cooperative oncogene and one of the central players in oncogenesis in many cancers. Altered c-Myc expression is often an early step in multistage transformation and one on which other mutations are based (Dang, 2012, Cell 149, 22-35). Therefore, there is an apparent addiction of cancer cells to de-regulated c-Myc, as proposed in 2008 by Weinstein. This Achilles heel offers a potential therapeutic window for cancer cells. c-Myc up regulation has been noted in most liquid and solid tumours with colon cancer forming one of the top hierarchies in c-Myc up regulation. ME1a1 binding site between P1 and P2 promoter of c-Myc is required for sustenance of transcriptionally active dual c-Myc promoters (Albert et al., 2001, J. Biol. Chem. 276, 20482-20490). Since the P2 promoter is associated with 75-90% of the c-Myc transcripts (Wierstra and Alves, 2008, Cancer Res. 99, 113-333), it serves as an ideal candidate for targeted therapy. We have previously demonstrated that siRNA against c-Myc could induce TGS in glioma cells, leading to increased cell death (Mehndiratta et al., 2011, Mol. Pharm. 8, 2302-2309).
The possibility of rendering cancer cells more sensitive to drugs or toxins by introducing “suicide genes” has two alternatives: toxin gene therapy, in which the genes for toxic products are transduced directly into tumour cells, and enzyme-activating prodrug therapy, in which the transgenes encode enzymes that activate specific prodrugs to create toxic metabolites. The latter approach is known as suicide gene therapy, gene-directed enzyme prodrug therapy (GDEPT) (Bridgewater et al., 1995 Eur. J. Cancer Oxf. Engl. 1990 31A, 2362-2370; Marais et al., 1996, Cancer Res. 56, 4735-4742), virus-directed enzyme prodrug therapy (VDEPT) (Huber et al., 1994, Ann. N. Y. Acad. Sci. 716, 104-114; discussion 140-143) or gene prodrug activation therapy (GPAT) (Eaton et al., 2001, Gene Ther. 8, 557-567), which could be utilized in isolation or combined with other strategies to make a significant impact on cancer treatment.
PLAP promoter is a type of tumour type specific promoter which was characterized by Deng et al in 1992 (Cancer Res. 52, 3378-3383). Most of the tumour specific promoters like alpha-fetoprotein, specific for hepatocellular carcinoma (HCC), are polymerase II driven (Peng et al., 2013, LoS ONE 8, e53072). Often, such tumour specific promoters are weak in nature (Qiao et al., 2002, Gene Ther. 9, 168-175). Full length PLAP promoter sequence spans from −512 bp to +24 that is about 536 bp long, but the region between −363 to −170 bp contains strong negative control elements (Deng et al., 1992b). Presence of this silencing region in the whole promoter leads to the decrease in promoter activity, therefore we selected the region between −170 and +24 bp. When we testified the promoter activity of this region, it has the necessary strength to drive a transgene specifically in variety of PLAP expressing tumour cell lines.
Placental Like Alkaline Phosphatase (PLAP) was one of the first proteins found to be ectopically expressed by cancer cells; leading to the concept that deregulation of embryonic genes plays a significant role in the cancer process (Fishman et al., 1968, Enzymologia 34, 317-321; Fishman et al., 1968, Nature 219, 697-699). PLAP or the Regan isoenzyme has been demonstrated in malignancies of the lung, testis, ovary, pancreas, colon, lymph tissue, kidney, stomach, and bladder. Placental-like alkaline phosphatase or GCAP or Nagao isoenzyme is most frequently expressed in germ cell tumours and in ovarian cancer and serves as a useful tumour marker in patients with those tumours (Loose et al., 1984, Am. J. Clin. Pathol. 82, 173-177). The highest level of elevation seems to comprise germ cell tumours of the testis (Nathanson and Fishman, 1971, Cancer 27, 1388-1397, Sasaki and Fishman, 1973, Cancer Res. 33, 3008-3018). In the case of seminomas, PLAP or PLAP like enzymes seem to be established as clinically useful tumour markers (Jeppsson et al., 1984, Int. J. Cancer J. Int. Cancer 34, 757-761; Lange et al., 1982, Cancer Res. 42, 3244-3247). As the assays cannot distinguish between the closely related tumour markers GCAP and PLAP and there is only 12 amino acid substitution between the two proteins, so from last 30 years PLAP is generally used as a tumour marker. (Szentirmay et al., 1982, Cancer Detect. Prev. 5, 185-194.), suggested that PLAP could also be an oncodevelopmental marker of human gastric neoplasia.
Placental Like Alkaline Phosphatase (PLAP) also known as Germ Cell Alkaline Phosphatase (GCAP) is a marker of cancers of the ovary, testis, lung, and the gastrointestinal tract. GCAP is a useful immune-histochemical marker of carcinoma in situ of the testis. Elevated levels of PLAP or PLAP-like alkaline phosphatases have been demonstrated for a number of different malignancies including those from pancreas (in 27-30% cases), lung (9-40%), breast (5-23%), colon (10-54%), lymph nodes, kidney, stomach (36%) and bladder. (Jeppsson et al., 1984, Int. J. Cancer 34, 757-761; Lange et al., 1982, Cancer Res. 42, 3244-3247)
PLAP is ectopically expressed in wide range of tumors and consequently its promoter is active only in such neoplastic transformations with little or no activity in normal/untransformed cells, so this promoter can be utilized for generating various tumour specific therapeutic modalities. (Milin, Jos Luis 1992, In Book entitled: Mammalian Alkaline Phosphatases, From Biology to Applications in Medicine and Biotechnology).
U.S. Pat. No. 6,867,036 relates to a nucleic acid construct which provides cell type-specific expression of a therapeutic transgene. In one embodiment, the amplification promoter element is a heat shock response element (HSE) and the transcription activator is HSF-1. The construct enables functional targeting of a therapeutic gene while avoiding undesirable effects in non-targeted cells, by combining sufficiently high-level expression to promote a desirable therapeutic outcome with exceptional tissue specificity. A series of promoter elements, constructs, vectors, and therapeutic approaches is presented for gene therapy of tumours such as melanoma and other genetic diseases.
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U.S. Pat. No. 7,321,030 relates to a promoter domain in the upstream side of exon 1B of IAI.3B gene has a specifically high promoter activity in ovarian cancer cells. An adenovirus having this promoter domain inserted in the E1 domain thereof exhibits a specifically high cell proliferation inhibitory effect on ovarian cancer cells. Thus, it is efficacious in gene therapy for ovarian cancer.
The majority of current therapeutic agents for cancer, in both cytotoxic and non cytotoxic categories, are chemicals foreign to the human body. Since most of these agents were designed by humans, not the nature, they have very high chances to bind to and interact with other cellular factors than their expected targets in the body. These “off-target” bindings and interactions account for significant opportunities for side effects. Radiation therapy causes severe epithelial damage leading to local necrosis. It can also cause radiation cystitis, sterility, hair loss, and fibrosis and generalized fatigue. Since radiation causes DNA damage it might cause secondary tumors also. Whatever be the therapy employed the greatest limitation of all the currently available cancer therapeutics is the inability to differentiate between normal and neoplastic cells.
The problem with the currently available cancer therapies is the lack of specificity of the same towards cancer cells, thus gives up rise to toxicity. Till now there has not been a efficient therapy which is able to combat wide range of tumours and induce cytotoxicity in neoplastically transformed cells only and spare the normal or untransformed cells.